The NF-kappaB/Rel family of transcription factors plays an important role in inflammatory responses. In nonstimulated cells, the heterodimeric NF-kappaB complex is located in the cytoplasm, where it is associated with the inhibitory molecule, IkappaBalpha. In response to stimulation by the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1, IkappaBalpha undergoes phosphorylation and subsequent proteolytic degradation, allowing the p50/p65 NF-kappaB heterodimer to migrate to the nucleus and transactivate various inducible target genes. The IkappaB kinase (IKK) complex plays a pivotal role in the phosphorylation of IkappaBalpha in response to cytokine stimulation. This complex is composed of at least two catalytic subunits, IKKalpha and IKKbeta, and a tightly associated regulatory subunit known as NEMO. A number of studies have established that the Ras superfamily of small GTP-binding proteins plays important roles in signal transduction, proliferation, and malignant transformation but also inflammation. Ras and some Ras-like proteins (e.g., RhoB, Rheb, TC10) require posttranslational modification by conjugation of a farnesyl (15-carbon isoprenyl group) moiety to the C-terminal. After farnesylation, these Ras proteins are localized to the inner surface of the plasma membrane, and become functional. Inhibitors of protein farnesylation have been shown to block nuclear targeting of NF-kappaB by oncogenic Ras; however, whether protein farnesylation plays a role in cytokine-dependent regulation of NF-kappaB is unknown. In this study, we examined the effect of manumycin A, a potent and selective farnesyltransferase inhibitor, on cytokine-induced NF-kappaB activation in CHO cells and human liver-derived HepG2 cells. Manumycin A has antitumor activity in vitro, and in vivo studies in nude mouse xenograft models has demonstrated little toxic side effects. To assess whether protein farnesylation may play a role in the induction of NK-kappaB by cytokines, cells were exposed to 10 uM manumycin A for periods up to 6 h. This inhibitor clearly blocked NF-kappaB transactivation and nuclear translocation of p65/RelA in response to TNF. Manumycin A also blocked IKK-dependent phosphorylation of IkappaB alpha elicited by TNF or interleukin-1 beta. Using solid-phase kinase assays, it was determined that activation by TNF of the IKK complex and an upstream kinase, NF-kappaB inducing kinase (NIK), was rapidly attenuated after cell treatment with manumycin A for 30-60 min. Moreover, ectopic expression of NIK or IKKbeta in HepG2 cells did not confer protection against manumycin A. These results indicate that farnesylated proteins with a short half-life are involved in the up-regulation of NF-kappaB through signaling mechanisms that control the IKK pathway. Using blue native gel electrophoresis, we found the ~750 kDa IKK complex to be stable in the presence of manumycin A, but the TNF-dependent recruitment of IKK to lipid rafts was lost, an event that was correlated with actin depolymerization. Therefore, it would appear that manumycin A interferes with the recruitment and/or formation of signaling-competent complexes at the plasma membrane upon cytokine stimulation in cells devoid of K-ras mutation. Our results indicate a novel link between manumycin-sensitive targets and IKK activation elicited by TNF. As proinflammatory cytokines play an important role in the pathogenesis of insulin resistance, our findings suggest that inhibition of cytokine signaling at a converging step at or upstream of IKK may represent a potential target for new strategies to improve insulin-resistant states.